基于G-四链体模板的银纳米簇共振能量转移比率检测miRNA  

FRET-based Ratiometric MicroRNA Detection with G-quadruplex-stabilized Silver Nanoclusters

作　　者：林若韵 陈阳 陶广宇 裴晓静[1] 刘锋[1] 李娜[1] 

机构地区：[1]北京分子科学国家实验室生物有机与分子工程教育部重点实验室北京大学化学与分子工程学院分析化学所,北京100871

出　　处：《化学学报》2017年第11期1103-1108,共6页Acta Chimica Sinica

基　　金：国家自然科学基金(Nos.21475004和21535006)资助~~

摘　　要：以DNA为模板合成的荧光银纳米簇(DNA-Ag NC)是一类性能优秀的发光探针,已被广泛应用于传感与成像领域.目前大多数定量方法都是基于目标物结合带来的直接的银纳米簇发光强度的变化,因此,分析方法的重现性将不可避免地受实验场地或测定条件变化的影响.本工作中,采用以G-四链体为模板的银纳米簇(GQ-AgNC)与菁染料Cyanine5(Cy5)为能量供-受体对,发展了基于荧光共振能量转移(FRET)机理的mi RNA比率测定方法.其中,Cy5标签具有通用性,简化了实际应用中的实验设计,降低了试剂成本.针对mi RNA let-7a的方法,检测动态范围为12~300nmol/L,检测限为6.9 nmol/L,且可以将let-7a与let-7家族中的其他mi RNA区分.HepG2细胞的总RNA提取物加标回收结果表明该方法具有应用于临床样本测试的潜力.本研究工作拓展了DNA-Ag NC的应用,并有助于加深在FRET设计中以DNA-Ag NC为供体进行分析应用的进一步理解.Fluorescent DNA-stabilized Ag nanoclusters（DNA-Ag NCs）, a class of excellent luminescence probes with excellent optical properties, have been applied in assorted sensing and imaging fields. To date, most of the quantifications were based on the direct signal change of DNA-Ag NCs caused by target recognition, which inevitably jeopardizes the reproducibility and robustness of methods when experimental settings or detecting conditions are changed. In this work, using the highly fluorescent G-quadruplex-stabilized Ag NCs（GQ-Ag NCs） and Cy5 as the donor-acceptor pair, we for the first time developed a FRET based ratiometric method for mi RNA detection. A rationally optimized hairpin recognition structure was attached to the G-quadruplex template of the Ag nanocluster. The introduction of target sequence opened the hairpin, led to the approximation of the donor nanocluster and the acceptor Cy5, enabled the energy transfer between the FRET pair, and thus generated the optical signal change. The Cy5 tag sequence was designed to be universal, simplifying the experimental design and reduced the cost in applications. The optical signal for quantitation was determined by the signal difference between the Ag nanocluster and the Cy5 fluorophore, with the fluorescence intensity of Cy5 used as internal reference in order to prevent signal variation. Micro RNAs（mi RNA） are short RNA molecules that have emerged as a kind of key post-translational regulators of gene expression in eukaryotic organisms. In this study, micro RNA let-7 a was chosen as the model target of our FRET-based ratiometric detection for demonstration. The linear range and the detection limit of the method on let-7 a was 12~300 nmol/L and 6.9 nmol/L, respectively. The proposed method presented reasonable selectivity amongst the members of the same let-7 family. The remarkable recovery in total RNA extracted from HepG2 cell lines demonstrated the potential in clinical applications. The highlights of our work extended the application of DNA-templated A

关 键 词：G-四链体 银纳米簇 荧光共振能量转移 比率检测 MIRNA 

分 类 号：O629.7[理学—有机化学] O657.3[理学—化学]